Cell identity and behavior are determined by the repertoire of genes that are expressed within the particular cell. Of approximately 26,000 genes that are present in the human genome, only a fraction is expressed within a particular cell type at a given time. Cells alter the pattern of gene expression in order to accommodate the changing environment. Multiple mechanisms are employed by the cell to regulate gene expression. For instance, regulatory sequences such as promoters and enhancers, which are usually found in untranslated regions of genes, contain elements that are specifically recognized by intracellular DNA-binding proteins that are called transcription factors (TFs). The binding of transcription factors to their cognate sites in a regulatory sequence controls the recruitment of the basal transcription machinery that initiates gene transcription. The ability of cis-regulatory sequences and transcription factors to activate transcription is referred to as the transcriptional activity. Gene expression is also known to be regulated at the post-transcriptional level. For example, many RNAs contain cis-regulatory elements that regulate the maturation, stability, and or degradation of RNA transcripts. The multiple mechanisms of regulating transcript levels allow cells to maintain the homeostatic regulation of gene expression.
Impairments of the finely tuned regulation can cause cell death, transformation, or metabolic diseases. Therefore, it is important to put in place technologies that enable the assessment of the activities of the machinery that controls gene expression.
For example, easily detected reporter proteins, such as luciferase, chloramphenicol acetyltransferase, and green fluorescent protein, can be made to be expressed by reporter constructs that are introduced into a cell. Detecting reporter proteins, however, is not well suited for assays intended to measure multiple transcriptional activities. This is due, in part, to the relatively small numbers of protein products amenable to convenient and rapid detection, and to the differences in transcribed amounts and stabilities between different reporter proteins.
Another broad class of approaches to assess gene expression is based on the evaluation of the amounts of gene transcripts, e.g., Northern blotting, reverse transcription PCR, and RNA array hybridization. However, as RNA abundance is regulated by many mechanisms, including the transcriptional regulation, RNA processing, accumulation, and degradation, these methods provide limited information about the activities of cis-regulatory sequences and trans-acting factors. Moreover, while the use of reporter construct libraries to detect multiple transcription factor activities have been described (see, e.g., U.S. patent application Publication Ser. Nos. 2003/0148287 and 2003/0143547), the problems associated detecting RNA abundance, including differences in the transcription levels, methylation, stability, hybridization efficacy, and susceptibilities to RNAses, that exist between different reporter RNAs expressed by reporter construct libraries, have not been addressed.
As transcription factors need to bind their cognate DNA sequence in order to initiate transcription, DNA binding has been widely used as a marker of transcription factor activation. Gel-shift assay, also known as EMSA, is a classical method for assessing DNA binding. However, many mechanisms exist that can effect the activity of transcription factors at multiple levels of regulation independently from DNA binding (Baldwin (2001) J Clin Invest 107:241-246). Therefore, DNA binding assays provide only limited information about the ability of transcription factors to activate transcription.
As impaired regulation of various transcription factors has been associated with various human diseases, including chronic inflammatory conditions, autoimmunity, and cancer (see, e.g., Makarov (2000) Mol Med Today 6:441-448; Baldwin et al. (2001) J. Clin Invest 107:241-246), transcription factors are considered attractive targets for drug development. To screen libraries of synthetic or biological compounds for their ability to selectively modulate the transcriptional activity of transcription factors of interest, one needs an appropriate assay enabling high throughput profiling of numerous transcription factors. To start addressing these questions, the art requires technology that permits the profiling of the activities of numerous transcription factors. The progress in this area has been hampered by the lack of adequate tools.